Biosynthesis of iso-fatty acids in myxobacteria

Article abstract of DOI:10.1039/b504889c

The fatty acid (FA) profiles of the myxobacteria Stigmatella aurantiaca and Myxococcus xanthus were investigated by acidic methanolysis of total cell extracts and GC or GC-MS analysis. The main components were 13- methyltetradecanoic acid (iso-15:0) and (Z)-hexadec-11-enoic acid (16:1, ω-5 cis). The biosynthesis of iso-FAs was investigated in several feeding experiments. Feeding of isovaleric acid (IVA) to a mutant impaired in the degradation of leucine to isovaleryl-CoA (IV-CoA) (bkd mutant) of M. xanthus only increased the amount of iso-odd FAs, whereas feeding of isobutyric acid (IBA) gave increased amounts only of iso-even FAs. In contrast, a bkd mutant of S. aurantiaca gave increased amounts of iso-odd and iso-even fatty acids in both experiments. We assumed that in S. aurantiaca α-oxidation takes place. [D₇]-15-Methylhexadecanoic acid (8) was synthesised and fed to S. aurantiaca as well as [D₁₀]leucine and [D₈]valine to elucidate this pathway in more detail. The iso-fatty acid 8 was degraded by α- and β-oxidation steps. [D₁₀]Leucine was strongly incorporated into iso-odd and iso-even fatty acids, whereas the incorporation rates for [D₈]valine into both types of fatty acids were low. Thus α-oxidation plays an important role in the biosynthesis of iso-fatty acids in S. aurantiaca. The incorporation rates observed after feeding of [D ₁₀]leucine and [D₈]valine are the highest for iso-17:0 compared to the other acids. This indicates the central role of iso-17:0 in the biosynthesis of iso-FAs. The shorter homologues seem to be formed mainly by α-oxidation and β-oxidation of this acid. After feeding of 8 traces of unsaturated counterparts of this labelled FA occurred in the extracts indicating that desaturases are active in the biosynthesis of unsaturated fatty acids in S. aurantiaca. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b504889c

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A R T I C L E
Biosynthesis of iso-fatty acids in myxobacteria†
Jeroen S. Dickschat,a Helge B. Bode,b Reiner M. Kroppenstedt,c Rolf Mu¨llerb and
Stefan Schulz*a
a Institut fu¨r Organische Chemie, TU Braunschweig, Hagenring 30, 38106 Braunschweig,
Germany. E-mail: stefan.schulz@tu-bs.de; Fax: +49 531 391 5272; Tel: +49 531 391 7353
b Institut fu¨r Pharmazeutische Biotechnologie, Universita¨t des Saarlandes, Geb. 8.1, 66123
Saarbru¨cken, Germany
c Deutsche Sammlung fu¨r Zellkulturen und Mikroorganismen (DSZM), Mascheroder Weg 1b,
38124 Braunschweig, Germany
Received 11th April 2005, Accepted 31st May 2005
First published as an Advance Article on the web 27th June 2005
The fatty acid (FA) profiles of the myxobacteria Stigmatella aurantiaca and Myxococcus xanthus were investigated by
acidic methanolysis of total cell extracts and GC or GC-MS analysis. The main components were
13-methyltetradecanoic acid (iso-15:0) and (Z)-hexadec-11-enoic acid (16:1, x-5 cis). The biosynthesis of iso-FAs was
investigated in several feeding experiments. Feeding of isovaleric acid (IVA) to a mutant impaired in the degradation
of leucine to isovaleryl-CoA (IV–CoA) (bkd mutant) of M. xanthus only increased the amount of iso-odd FAs,
whereas feeding of isobutyric acid (IBA) gave increased amounts only of iso-even FAs. In contrast, a bkd mutant of
S. aurantiaca gave increased amounts of iso-odd and iso-even fatty acids in both experiments. We assumed that in S.
aurantiaca a-oxidation takes place. [D7]-15-Methylhexadecanoic acid (8) was synthesised and fed to S. aurantiaca as
well as [D10]leucine and [D8]valine to elucidate this pathway in more detail. The iso-fatty acid 8 was degraded by
a- and b-oxidation steps. [D10]Leucine was strongly incorporated into iso-odd and iso-even fatty acids, whereas the
incorporation rates for [D8]valine into both types of fatty acids were low. Thus a-oxidation plays an important role in
the biosynthesis of iso-fatty acids in S. aurantiaca. The incorporation rates observed after feeding of [D10]leucine and
[D8]valine are the highest for iso-17:0 compared to the other acids. This indicates the central role of iso-17:0 in the
biosynthesis of iso-FAs. The shorter homologues seem to be formed mainly by a-oxidation and b-oxidation of this
acid. After feeding of 8 traces of unsaturated counterparts of this labelled FA occurred in the extracts indicating that
desaturases are active in the biosynthesis of unsaturated fatty acids in S. aurantiaca.
investigations were performed on the two species Stigmatella
Introduction
aurantiaca and Myxococcus xanthus, the fatty acid pattern
Myxobacteria are of considerable interest because of their
of which had previously been investigated briefly by Ware
complex life cycle, unusual physiology, and broad metabolic
and Dworkin and Schro¨der and Reichenbach, respectively.13,14
potential.1,2 During the last decades several compounds with
Furthermore, the fatty acid pattern of M. xanthus cells is
pharmacological activity have been isolated from different
highly dependent on the growth medium.15 Recently, the bound
species such as Myxococcus fulvus,3 Myxococcus xanthus,4
fatty acids in phosphatidylethanolamine (PE) purified from
Stigmatella aurantiaca,5 and Sorangium cellulosum.6 The biosyn-
M. xanthus cell membranes have been identified.16
thesis of their metabolites often comprises unusual biosyn-
In a previous communication we reported on the biosynthesis
thetic mechanisms.7,8 One example is the unexpected effect of
bkd (branched chain keto acid dehydrogenase) mutagenesis in
M. xanthus and S. aurantiaca; bkd mutants are impaired in the
of iso-fatty acids (iso-FA) in M. xanthus and S. aurantiaca,
including results from genetically modified strains.17 The iso-
FAs with an odd number of carbon atoms (iso-odd FAs) were
degradation of the branched chain amino acids leucine, valine,
shown to be generated from the leucine-derived starter IV–
and isoleucine to isovaleryl-CoA (IV–CoA), isobutyryl-CoA
(IB–CoA), and 2-methylbutyryl-CoA (2MB–CoA), respectively.
In streptomycetes, a disruption of the bkd locus leads to a
CoA, while the iso-even FAs were derived from iso-odd FAs by
a-oxidation, a new pathway to methyl-branched FAs. The valine-
derived starter isobutyryl-CoA (IB–CoA) was not involved in
total depletion of the cells of any of these thioesters (and
the biosynthesis of these FAs. In the present article we will
therefore also of iso-fatty acids and secondary metabolites that
present the synthesis of the labelled key component 8. The
biosynthesis of iso-even FAs by a-oxidation of iso-odd FAs in
S. aurantiaca was clearly shown by the degradation of [D7]-
15-methylhexadecanoic acid (8) to iso-even FAs and iso-odd
require these thioesters as starting units).9,10 However, in the
two myxobacteria only a reduction of branched chain keto
acid dependent secondary metabolite formation and iso-fatty
acid biosynthesis is observed. The reason for this is an alter-
FAs with shorter chain length. Furthermore, full details on
native pathway to synthesise at least IV–CoA and presumably
the fatty acid composition of the investigated strains used in
also IB–CoA from acetyl-CoA. This novel pathway branches
feeding experiments and further conclusions on the fatty acid
from hydroxymethylglutaryl-CoA, a central intermediate of the
biosynthesis pathways that operate in these myxobacteria are
mevalonate dependent isoprenoid biosynthesis.11,12 In the course
presented.
of further studies regarding this pathway and its connection
to fatty acid biosynthesis in general, we became interested in
whether the fatty acid biosynthesis in myxobacteria follows
established pathways or shows differences to other bacteria. The
Results and discussion
Extracts of liquid cultures of S. aurantiaca and M. xanthus
(wild-type strains and bkd mutants) were methanolysed under
acidic conditions and analysed by GC–MS. Identifications of
the resulting methyl esters of free and bound fatty acids were
† Electronic supplementary information (ESI) available: Further details.
See http://dx.doi.org/10.1039/b504889c
2 8 2 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 2 4 – 2 8 3 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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based upon mass spectra18,19 and gas chromatographic retention
indices (see ESI†).20,21
acid furthermore plays a central role in the FA metabolism of
S. aurantiaca, because all other iso-FAs with shorter chain length
are at least partially generated from this acid via a- and/or
b-oxidation steps. Compound 8 was prepared starting with 4-
bromobutyric acid 1 that was reduced to the corresponding
alcohol 2 using borane-dimethyl sulfide (Scheme 1).26,27 The
deuterium label and the methyl branch were introduced in a
cross coupling reaction with [D7]isopropylmagnesium bromide
catalysed by Li2[CuCl4],28 after protection of the alcohol with
dihydropyran.29 Deprotection furnished [D7]-6-methylhexan-1-
ol which was transformed into the corresponding aldehyde 5
by PCC oxidation.30,31 This aldehyde was immediately used in
a Wittig reaction with 10-hydroxydecyltriphenylphosphonium
ylide prepared from 2-(10-bromodecyloxy)oxane.32 Unexpect-
edly, the protecting group was lost during the formation of the
phosphonium salt with triphenylphosphane. The obtained un-
saturated alcohol [D7]-(Z)-15-methylhexadec-10-en-1-ol 7 was
hydrogenated using Pd/C to give [D7]-15-methylhexadecan-1-
ol, and then oxidised with PDC in DMF to yield 8.33 The results
of the feeding experiments with S. aurantiaca are summarised in
Table 1 and the total ion chromatogram is presented in Fig. 1.
The fatty acid profiles of both species are summarised in
Tables S1 and S2 of the ESI, respectively†. S. aurantiaca cells
contained a number of saturated and unsaturated unbranched
FAs (C10–C18). The main component of this class was (Z)-
hexadec-11-enoic acid (16:1, x-5 cis, 24.0% of total extract).
Some saturated and unsaturated iso-FAs were also present (C11–
C17), predominated by 13-methyltetradecanoic acid (iso-15:0,
22.3%) and 15-methylhexadecanoic acid (iso-17:0, 11.9%). In
addition, 2-hydroxy and 3-hydroxy FAs (iso-17:0 3OH, iso-
15:0 3OH, and 16:0 2OH) were present in minor amounts.
Fautz et al. already reported on these hydroxy fatty acids as
characteristic lipid constituents of S. aurantiaca (strain Sg a1)
and other gliding bacteria.22 The fatty acid profile of other
myxobacteria also comprises hydroxy fatty acids.23 In contrast,
the bkd mutant which is impaired in the generation of methyl-
branched starting units from branched amino acids produced
significantly reduced relative amounts of branched FAs, whereas
the relative amounts of unbranched FAs increased, e.g. iso-15:0
(11.9%) and 16:1 (x-5 cis, 32.4%).
This pattern was repeated in M. xanthus. The fatty acid profile
consisted of saturated and unsaturated unbranched (C10–C17)
and methyl-branched (C13–C17) FAs with 16:1 (x-5 cis, 13.8%)
and iso-15:0 (37.4%) as major components. 2-Hydroxy and 3-
hydroxy FAs (iso-17:0 3OH, iso-17:0 2OH, iso-15:0 3OH, 8:0
3OH, and 16:0 3OH) were also found in low amounts. In the
bkd mutant the relative amounts of iso-FAs decreased compared
to unbranched FAs, reflected by 16:1 (x-5 cis) (27.6%) and iso-
15:0 (14.6%). This decrease of iso-FAs in combination with an
increase of unbranched unsaturated FAs in the bkd mutant of
M. xanthus was reported earlier.12,15,16,24
In earlier investigations on M. xanthus and S. aurantiaca
among the unbranched FAs only C11–C17 (M. xanthus) and C12–
C17 (S. aurantiaca) have been detected, whereas iso-FAs ranged
from C14–C17 (M. xanthus) and C13–C17 (S. aurantiaca), respec-
tively, with iso-15:0, iso-17:0, and 16:1 as main components
in the fatty acid extracts of both species.13,14 These FAs also
predominate in other myxobacteria.23,25
In contrast to a total depletion of the cells of any iso-branched
fatty acids after disruption of the bkd locus as observed in other
bacteria (e.g. Streptomyces),9,10 myxobacteria have an alternative
pathway to IV–CoA that branches from the mevalonate depen-
dent isoprenoid biosynthesis.11,12 This pathway is induced in the
bkd mutants and gives rise to residual iso-FAs.
In order to elucidate the biosynthetic pathways to branched
FAs we fed the bkd mutants with the methyl-branched acids
isobutyric acid (IBA) and isovaleric acid (IVA) known to be
formed by the degradation of valine and leucine, respectively
(Tables S1 and S2 in the ESI†). The biosynthesis of iso-even
FAs normally takes place by chain elongation of IB–CoA with
acetate-derived malonyl-CoA, while iso-odd FAs arise from
elongation of IV–CoA. We obtained significantly increased
amounts only for the expected FAs after feeding the bkd mutant
of M. xanthus with IBA or IVA (e.g. increased amounts of
14-methylpentadecanoic acid, iso-16:0, after feeding of IBA,
and increased amounts of 13-methyltetradecanoic acid, iso-
15:0, after feeding of IVA, respectively), whereas the addition
of either IBA or IVA increased the amounts of both iso-odd and
iso-even FAs in the bkd mutant of S. aurantiaca. Therefore we
concluded that fatty acid biosynthesis in S. aurantiaca proceeds
with the involvement of a-oxidation of long chain fatty acids or
transformation of IBA to IVA and vice versa (see below), whereas
the respective pathway should not be active in M. xanthus.
The labelled key compound [D7]-15-methylhexadecanoic acid
8 ([D7]-iso-17:0) was fed to S. aurantiaca to further investigate
the participation of a-oxidation in the biosynthesis of iso-FAs.
This experiment is particularly important because it gives the
unambiguous proof for the formation of iso-even FAs from
iso-odd FAs by a-oxidation. As will be shown below, this
Scheme 1 Synthesis of [D7]-15-methylhexadecanoic acid (8). a) BH3–
SMe2, THF, 74%; b) 3,4-dihydro-2H-pyran, p-TsOH, Et2O, 91%;
c) [D7]-iPrMgBr, Li2[CuCl4], THF, 52%; d) p-TsOH, MeOH, 64%;
e) PCC, CH2Cl2, 53%; f) BuLi, THF, 45%; g) H2, Pd/C, Et2O, 78%;
h) PDC, DMF, 67%.
The iso-odd FAs occurred in significantly higher amounts
than the iso-even FAs. Incorporation of the deuterium label
originating from 8 (for the mass spectrum of its methyl ester
see Fig. 2A) is shown for all iso-FAMEs obtained after acidic
methanolysis by the occurrence of peaks that elute directly
before the unlabelled FAMEs in GC. These labelled methyl
esters are readily identified by their mass spectra. Molecular
ions are increased by seven units, e.g. from m/z = 270 to 277
in the case of methyl 14-methylpentadecanoate derived from
iso-16:0 (compare Figs. 2B and C). Furthermore, some high
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 2 4 – 2 8 3 1
2 8 2 5

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Table 1 Fatty acid profile of Stigmatella aurantiaca after feeding of
8. Fatty acids were identified as methyl esters in a methylated fatty
acid extract. Peak: peak marker in Fig. 1, trace compounds are not
marked (—), I: retention index, Int.: percent of total area in GC, Inc.:
incorporation rate of deuterium labelling in labelled compounds
spectra of deuterated compounds show a decreased fragment
ion at m/z = 43 in combination with an increased signal at
m/z = 50. Both effects are in agreement with the [D7]isopropyl
group present in iso-FAMEs. The same changes in mass
spectra from unlabelled to labelled compounds can be observed
for methyl 13-methyltetradecanoate (obtained from iso-15:0,
Figs. 2D and E), methyl 12-methyltridecanoate (iso-14:0), and
methyl 11-methyldodecanoate (iso-13:0), respectively (data not
shown). The incorporation of deuterium label into methyl 14-
methylpentadecanoate (iso-16:0) proves that iso-17:0 must be
degraded by a-oxidation in S. aurantiaca. The occurrence of
the deuterium label in methyl 13-methyltetradecanoate (iso-
15:0) shows that b-oxidation also takes place, or, less likely,
double a-oxidation. The chain lengths of methyl-branched
FAMEs vary from methyl 9-methyldecanoate (iso-11:0) to
methyl 14-methylpentadecanoate (iso-16:0, with missing methyl
10-methylundecanoate, iso-12:0), and incorporation rates rise
from 33% for methyl 12-methyltridecanoate (iso-14:0) to 58% in
case of methyl 14-methylpentadecanoate (iso-16:0). An increas-
ing incorporation rate depending on the chain length in both
series of iso-even and iso-odd FAs can be observed (Table 1).
Furthermore, incorporation into 2-hydroxy and 3-hydroxy
FAs is shown by the presence of the deuterated analogs
of methyl 2-hydroxy-15-methylhexadecanoate (iso-17:0 2OH)
and methyl 3-hydroxy-15-methylhexadecanoate (iso-17:0 3OH),
respectively. The mass spectra of labelled and unlabelled methyl
2-hydroxy-15-methylhexadecanoate (iso-17:0 2OH) are shown
in Figs. 2F and G. The molecular ion indicates an incorporation
of seven deuterium atoms by an increase from m/z = 300 to
307. Other fragment ions shift from m/z = 222, 241, and 268 to
m/z = 229, 248, and 275. The occurrence of a base peak at m/z =
50 in the spectrum of the deuterated compound clearly indicates
the position of the deuterium atoms in the isopropyl moiety.
This pattern is repeated in the mass spectra of labelled and
unlabelled methyl 3-hydroxy-15-methylhexadecanoate (iso-17:0
3OH, Figs. 2H and I). The fragment ion at m/z = 103 resulting
from 3,4-cleavage next to the alcohol function, characteristic of
3-hydroxy fatty acid methyl esters,34 does not shift, and this is in
agreement with the location of labelling.
Peak
Compound
I
Int.
0.04
Inc. (%)
36
—
—
—
a
b
c
d
e
f
g
h
i
j
k
10:0
[D7]iso-11:0
iso-11:0
11:0
12:0
[D7]iso-13:0
iso-13:0
13:0
[D7]iso-14:0
iso-14:0
14:1 (x-5 cis)
14:0
[D7]iso-15:1
iso-15:1
[D7]iso-15:0
iso-15:0
15:1
15:0
[D7]iso-16:0
iso-16:0
1333
1390
1395
1432
1526
1583
1588
1626
1682
1689
1715
1726
1765
1770
1783
1789
1820
1824
1882
1887
1914
1925
1935
1940
1963
1971
1985
1990
2026
2048
2080
2091
2098
2103
2109
2124
2136
2142
0.04
0.05
0.18
0.91
0.18
0.14
0.15
0.09
0.17
0.12
0.67
0.10
0.13
5.42
5.00
0.07
0.15
2.32
1.68
3.64
2.29
0.08
0.85
1.31
1.12
54
33
44
58
l
m
—
—
n
o
p
q
—
r
s
t
58
10
16:1 (x-5 cis)
16:0
[D7]iso-15:0 3OH
iso-15:0 3OH
[D7]iso-17:1a
[D7]iso-17:1a
[D7]iso-17:0
iso-17:0
u
49.3
—
—
—
v
—
—
—
w
—
—
x
2.34
0.08
0.25
0.26
0.07
0.07
0.25
1.67
0.20
0.10
0.68
17:0
16:0 2OH
16:0 3OH
18:2
18:1
[D7]iso-17:0 2OH
iso-17:0 2OH
18:0
[D7]iso-17:0 3OH
iso-17:0 3OH
13
13
Besides the branched and unbranched saturated compounds
some unsaturated FAMEs were readily identified from their
mass spectra in the extracts obtained after feeding of 8. For the
determination of the double bond positions derivatisation with
dimethyl disulfide (DMDS) was performed.35 The mass spectra
of DMDS derivatives show strong fragmentation between the
methylthio groups, and the fragment ion containing the methyl
ester function is characterised by the loss of methanol (Figs. S1
and S2 and Table S3 in the ESI†). Interestingly, the unbranched
unsaturated fatty acids produced by S. aurantiaca are 16:1 (x-5
cis) and 14:1 (x-5 cis), respectively. The latter one can arise from
a Partially coeluting [D7]iso-17:1 (x-4 cis, x-5 cis, x-6 cis, and x-7 cis)
as determined from also partially coeluting DMDS derivatives.
mass fragment ions shift from m/z = 213, 227, 239, and 241
to m/z = 220, 234, 246, and 248, respectively, all indicating
the incorporation of seven deuterium atoms. Notably, fragment
ions characteristic of methyl esters (m/z = 74 and 87) do
not shift because the deuterium label is not located in the
functional group moiety of the FAMEs. Furthermore, mass
Fig. 1 Total ion chromatogram of an methylated fatty acid extract of Stigmatella aurantiaca after feeding of 8. Letters refer to compounds in Table 1.
Non-fatty acids are indicated by asterisks.
2 8 2 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 2 4 – 2 8 3 1

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Fig. 2 Mass spectra of labelled and unlabelled fatty acid methyl esters occurring in the methylated fatty acid extract of Stigmatella aurantiaca
after feeding with [D7]-15-methylhexadecanoic acid (8). [D7]-Methyl 15-methylhexadecanoate, [D7]-iso-17:0, peak u in Fig. 1 (A), [D7]-methyl
14-methylpentadecanoate, [D7]-iso-16:0, peak n (B), methyl 14-methylpentadecanoate, iso-16:0, peak o (C), [D7]-methyl 13-methyltetradecanoate,
[D7]-iso-15:0, peak l (D), methyl 13-methyltetradecanoate, iso-15:0, peak m (E), [D7]-methyl 2-hydroxy-15-methylhexadecanoate, [D7]-iso-17:0
2OH (F), methyl 2-hydroxy-15-methylhexadecanoate, iso-17:0 2OH, peak w (G), [D7]methyl 3-hydroxy-15-methylhexadecanoate, [D7]-iso-17:0 3OH
(H), methyl 3-hydroxy-15-methylhexadecanoate, iso-17:0 3OH, peak x (I).
16:1 (x-5 cis) through one b-oxidation step. Double a-oxidation
is unlikely, because 15:1 is only present in trace amounts. The
double bond position and configuration of 16:1 (x-5 cis) was
previously not identified in investigations on S. aurantiaca. Fautz
et al. reported on the presence of a hexadecenoic acid in this
species which they assumed to be 16:1 (x-5 cis) because of its
retention time relative to 16:1 (x-7 cis).22 Unsaturated 16:1 (x-5
cis) has also been identified in M. xanthus.15,16
Methyl-branched unsaturated FAs were only produced in
minute amounts and they all showed the labelling pattern of
8. The position of the double bonds in the labelled methyl esters
obtained from iso-17:1 varies from x-7 to x-4, also determined
from DMDS derivatives (Table S3 in the ESI†). The correspond-
ing free acids were only found in extracts of cultures fed with
8. The incorporation of labelling into the unsaturated iso-FAs
indicates that they arise from 8 by the action of a desaturase, and
this might also be the case for the major unsaturated acids 16:1
(x-5 cis) and 14:1 (x-5 cis), respectively. Bacteria can synthesise
unsaturated fatty acids by introduction of the double bond
during chain assembly (anaerobic pathway), while eukaryotes
generally introduce double bonds after chain assembly into
the saturated hydrocarbon chain requiring NAD(P)H and O2
(aerobic pathway) by action of a desaturase.36 Nevertheless,
such desaturases are also known from prokaryotes as Bacillus
subtilis.37–41
In subsequent experiments we tested whether the degradation
of long chain iso-odd FAs by a-oxidation is the only pathway to
iso-even FAs or the alternative route, transformation of valine
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 2 4 – 2 8 3 1
2 8 2 7

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Table 2 Fatty acid profile of Stigmatella aurantiaca after feeding of
[D8]valine and [D10]leucine, respectively. Fatty acids were identified as
methyl esters in a methylated fatty acid extract. I: retention index, Int.:
percent of total area in GC after feeding of [D8]valine and [D10]leucine,
respectively. Incorporation rates are given in percent in parentheses
Compound
I
Int. [D8]Val
0.42
Int. [D10]Leu
10:0
1333
1388
1390
1395
1526
1582
1583
1588
1682
1683
1689
1715
1726
1781
1783
1789
1820
1824
0.82
0.04 (29)
[D9]iso-11:0
[D7]iso-11:0
iso-11:0
0.00 (0)
0.10
0.15
0.10
0.11
0.10 (33)
12:0
[D9]iso-13:0
[D7]iso-13:0
iso-13:0
[D9]iso-14:0
[D7]iso-14:0
iso-14:0
14:1 (x-5 cis)
14:0
[D9]iso-15:0
[D7]iso-15:0
iso-15:0
0.00 (0)
0.43
0.20
0.21 (32)
0.00 (0)
0.89
0.55
0.44
0.19
1.59
7.63 (35)
1.63
0.40 (2)
20.0
0.40
14.1
15:1
15:0
1.81
[D9]iso-16:0
[D7]iso-16:0
iso-16:0
16:1 (x-5 cis)
16:0
[D9]iso-17:0
[D7]iso-17:0
iso-17:0
2.64 (32)
1882
1887
1914
1925
1983
1985
1990
2026
0.44 (3)
13.5
16.6
5.50
23.4
7.35
16.8
1.71 (40)
0.34 (4)
8.81
0.62
2.59
0.00
17:0
formation can dilute the original label. Two different pathways
are therefore important in the degradation of iso-FAs. The
first one starts with iso-17:0 (40% incorporation) and proceeds
through subsequent b-oxidation steps, affording iso-15:0 (35%),
iso-13:0 (33%), and iso-11:0 (29%), respectively (Scheme 2).
The second degradation pathway from iso-17:0 starts with an
initial a-oxidation step forming iso-16:0 (32%) and successive
b-oxidation to iso-14:0 (32%). Thus the normal biogenesis of
iso-odd FAs from leucine as well as the unusual formation of
iso-even FAs from this amino acid through one a-oxidation and
subsequent b-oxidation steps is demonstrated. Transfer between
these pathways might still be possible by a-oxidation of acids
other than iso-17:0. This acid is the key component for the
biosynthesis of iso-FAs. The lower homologues iso-15:0, iso-
13:0, and iso-11:0 are preferentially liberated in the catabolic
process, but not at all or only in lower amounts during the
anabolic process leading to iso-17:0. Liberation during the latter
process would result in a decrease of the incorporation rate with
longer chain length.
Scheme 2 Biosynthetic pathways from leucine and valine to iso-FAs.
a: a-oxidation, b: b-oxidation.
If the other possibility for the formation of iso-even FAs,
the use of the starter IB–CoA arising from valine, was active,
high incorporation after feeding S. aurantiaca with [D8]valine
would be expected. However, only low incorporation rates were
observed, the highest being 4% in the case of [D7]iso-17:0.
Two other fatty acids contained the deuterium label, iso-16:0
(3%) and iso-15:0 (2%), whereas incorporation into shorter x-
1 methyl-branched FAs could not be found, probably due to
the low amounts of these acids in the extract. The reduced
labelling in iso-16:0 and iso-15:0 compared to iso-17:0 indicates
that the latter one might be their precursor, formed through one
a-oxidation or one b-oxidation step, respectively. An anabolic
pathway may also contribute to these acids.
The strongly reduced incorporation rates after feeding of
[D8]valine compared to [D10]leucine clearly indicate that the
valine-derived possible starter unit IB–CoA has not the same
if any relevance as leucine derived IV–CoA. Furthermore, the
highest incorporation after application of [D8]valine was found
for iso-17:0 that should arise from leucine degradation and chain
to the starter IB–CoA followed by elongation with malonyl-
CoA, is also active (Scheme 2). [D8]Valine and [D10]leucine,
respectively, were fed to S. aurantiaca and the incorporation rates
into the fatty acids were compared. The results are summarised
in Table 2. Addition of [D10]leucine to the bacterial cultures
led to the formation of iso-FAs carrying nine deuterium atoms
due to the loss of one deuterium in the transamination reaction
to [D9]-a-ketoisocaproic acid. Oxidative decarboxylation then
forms [D9]IV–CoA, the methyl-branched starting unit usually
used for the biosynthesis of iso-odd FAs. The fatty acid pattern
of S. aurantiaca obtained after feeding of [D10]leucine was
characterised by the presence of [D9]iso-17:0 that showed the
highest incorporation rate (40%) among the iso-FAs (Table 2).
All other labelled iso-FAs had a shorter chain length and lower
incorporation rates. Therefore these fatty acids seem to be
generated by the degradation of [D9]iso-17:0. The degradation
pathways can be reconstructed based on the incorporation rates,
because it is reasonable that each additional biosynthetic trans-
2 8 2 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 2 4 – 2 8 3 1

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elongation of the respective starter IV–CoA with six malonyl-
CoA units. Therefore, two pathways for the biosynthesis of iso-
FAs from valine can be discussed. The first is initiated by the for-
mation of an IB–CoA starter from valine, chain elongation with
seven malonyl-CoA units to yield iso-18:0, and final degradation
through a- and b-oxidation steps to furnish the shortened iso-
FAs. This pathway is unlikely because of the low incorporation
rates compared to feeding of [D10]leucine. Furthermore, the
required valine derived precursor for iso-17:0, iso-18:0, is not
present in the fatty acid extract. It seems more likely that valine
is funnelled into the leucine metabolism by transamination to
a-ketoisovalerate and the well-known subsequent transforma-
tion to the leucine precursor a-ketoisocaproate42 that is widely
distributed among diverse bacteria43 (Scheme 2). This a-keto
acid then serves as a precursor of IV–CoA as a starter in
branched chain fatty acid biosynthesis as discussed above. The
clearly reduced incorporation rates for [D8]valine compared to
[D10]leucine as well as the highest incorporation rate observed
for typically leucine derived iso-17:0 are in accordance with this
pathway.
Even-numbered unbranched FAs are normally built up
through an acetate starter unit and chain elongation with
malonyl-CoA units, while the use of propionate starters fur-
nishes odd-numbered FAs, normally a disfavoured process.36,44
In both investigated myxobacteria we obtained a typical pattern
that showed significantly higher amounts of unbranched even-
numbered FAs than odd-numbered ones. Whether a-oxidation
is involved in the formation of unbranched odd from even FAs in
myxobacteria and a fatty acid as e.g. 16:0 has a similar role as iso-
17:0 as precursor for shorter as well as unsaturated FAs remains
to be investigated. Fatty acid degradation by a-oxidation has
been described already in 1959 by Martin and Stumpf in peanuts
(palmitic acid),45 in higher plants,46 in humans,47 and in the
bacterium Sphingomonas paucimobilis.48 The a-oxidation has
also been found in the phytanic acid degradation pathway.49 The
methyl group at C-3 of phytanic acid blocks its catabolism by
b-oxidation. Instead an a-oxidation step leads to pristanic acid
that can be readily degraded by subsequent b-oxidation steps
furnishing propionate and acetate units, respectively.
glassware in a N2 atmosphere. Thin layer chromatography was
carried out using 0.2 mm pre-coated plastic sheets Polygram
Sil G/UV254 (Marcherey-Nagel GmbH & Co. KG, Du¨ren,
Germany). 1H NMR spectra were recorded on a Bruker AC200
(200 MHz) spectrometer with TMS as an internal standard.
J values are given in Hz. 13C NMR spectra were obtained using
a Bruker AC200 (50 MHz) with TMS as internal standard. Col-
umn chromatography was carried out using Merck Kieselgel 60.
Feeding experiments
The strains Stigmatella aurantiaca DW4/3-1 and EBS7-11 (bkd
mutant), and Myxococcus xanthus DK1622 and JD300 (bkd
mutant), their cultivation and generation of fatty acid methyl
esters have previously been described.12,20,24
Feeding experiments were performed as previously
described.11,12 IVA, IBA, [D10]leucine, or [D8]valine (1 mM each)
were added to 50 ml cultures of the strains in 250 ml Erlenmeyer
flasks. All strains were cultivated on a rotary shaker at 30 ◦C
and cells were harvested by centrifugation. The labelled fatty
acid 8 was dissolved in CHCl3 (0.2 M) and 100 ll were added
on a CTT-agar plate to a final concentration of 6.5 mM. After
evaporation of the solvent under sterile conditions, 300 ll of cells
were added on this spot and also allowed to evaporate prior to
incubation at 30 ◦C for four days.
Derivatisation with DMDS
As described by Leonhardt and de Vilbiss,35 freshly distilled
dimethyl disulfide (DMDS, 0.1 cm3) and a solution of iodine
(0.05 cm3, 5% in diethyl ether) were added to the methylated fatty
acid extract of Stigmatella aurantiaca (0.05 cm3). The reaction
mixture was kept for 12 h at 50 ◦C. Then a saturated solution of
Na2SO3 (0.02 cm3) was added to remove the iodine. The mixture
was diluted with diethyl ether (1 cm3) and dried with Na2SO4.
The volatiles were removed at 40 ◦C in a gentle stream of N2
and the residue was diluted with 0.05 cm3 of diethyl ether. The
obtained solution was immediately used for GC–MS analysis.
GC–MS
GC–MS analyses were carried out on a HP 6890 Series GC
System connected to a HP 5973 Mass Selective Detector
(Hewlett-Packard Company, Wilmington, USA) fitted with a
BPX-5 fused-silica capillary column (25 m × 0.22 mm id,
0.25 lm film, SGE Inc., Melbourne, Australia). Conditions were
as follows: inlet pressure: 77.1 kPa, He 23.3 mL min−1; injection
volume: 1 lL; transfer line: 300 ◦C; electron energy: 70 eV. The
Conclusions
The data obtained by feeding of labelled valine and leucine
as well as 8 support the described mechanisms, especially the
uncommon a-oxidation mechanism used by S. aurantiaca. Both
organisms, S. aurantiaca and M. xanthus, lack the capability
to produce IB–CoA from valine under the conditions tested.
In M. xanthus this is demonstrated by the fact that the wild-
type produces no iso-even FAs, whereas feeding the bkd mutant
with IBA leads to an increased formation of this type of fatty
acids. It remains to be elucidated on which pathways the bkd
mutant of M. xanthus is able to produce minute amounts of
iso-even FAs. Probably a second bkd complex enabling IB–CoA
production is induced in the bkd mutant. The low incorporation
rates for [D8]valine into iso-FAs indicate that S. aurantiaca is
not able to produce fatty acids via IB–CoA. In contrast to
M. xanthus the diversity of FAs in this species is broadened by
a-oxidation giving access to iso-even FAs that are thus derived
from IV–CoA and not from IB–CoA. Further studies will show
whether the a-oxidation is more common in the biosynthesis of
fatty acids in nature.
◦
GC was programmed as follows: 5 min at 50 C increasing at
5
◦C min−1 to 300 ◦C, and operated in splittless mode (60 s
valve time). The carrier gas was He at 1 cm3 min−1. Retention
indices I were determined from a homologous series of n-alkanes
(C7–C25).
Preparation of 4-bromobutan-1-ol 226
A solution of 1 (4.18 g, 25 mmol) in THF (10 cm3) was added
dropwise at 0 ◦C to a solution of borane dimethylsulfide complex
(2.28 g, 30 mmol, 3.0 cm3) in dry THF (20 cm3). The reaction
mixture was stirred overnight at room temperature and then
quenched by the addition of ethanol (5 cm3) and water (20 cm3).
The crude product was obtained by extraction with pentane
(3 × 50 cm3), drying with MgSO4, and removing of the solvents
in vacuo. Purification of the residue by column chromatography
on silica gel with pentane–diethyl ether (3 : 1) afforded 2 (2.81 g,
74%) as a colourless liquid. TLC (pentane–diethyl ether = 3 :
1): RF 0.18; GC: I 1033; dH(400 MHz; CDCl3; Me4Si) 1.65–1.81
(2 H, m, CH2), 1.83–2.04 (2 H, m, CH2), 3.46 (2 H, t, J 6.6,
CH2) and 3.70 (2 H, t, J 6.3, CH2); dC(100 MHz; CDCl3; Me4Si)
29.1 (CH2), 30.9 (CH2), 33.6 (CH2) and 61.8 (CH2); m/z (EI)
136 (36%), 134 (36), 108 (10), 106 (10), 95 (5), 93 (5), 73 (14), 55
(100) and 41 (38).
Experimental
Synthesis
General methods. Chemicals were purchased from Fluka
Chemie GmbH (Buchs, Switzerland) or Sigma-Aldrich Chemie
GmbH (Steinheim, Germany) and used without further purifica-
tion. Solvents were purified by distillation and dried according to
standard methods. All reactions were carried out in oven-dried
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 2 4 – 2 8 3 1
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Preparation of THP ethers
by column chromatography with pentane–diethyl ether (5 : 1)
yielded [D7]-5-methylhexan-1-ol (238 mg, 64%) as a colourless
liquid. TLC (pentane–diethyl ether = 5 : 1): RF 0.15; GC: I 942;
dH(200 MHz; CDCl3; Me4Si) 1.16–1.37 (4 H, m, 2 × CH2), 1.48–
1.64 (2 H, m, CH2), 1.81 (1 H, br s, OH) and 3.63 (2 H, t, J 6.6,
CH2); dC(50 MHz; CDCl3; Me4Si) 23.5 (CH2), 33.0 (CH2), 38.7
(CH2) and 62.9 (CH2); m/z (EI) 104 (5%), 87 (38), 77 (100), 62
(92), 50 (81) and 46 (89).
Similar to the method of Parham and Anderson,29 a solu-
tion of 2 or 10-bromodecan-1-ol, respectively, (1.0 equiv.),
3,4-dihydro-2H-pyran (1.1 equiv.) and a catalytic amount of
p-toluenesulfonic acid (5 mol%) in dry diethyl ether (final
concentration of the alcohol was approximately 5 mol dm−3)
was stirred at room temperature for 3 h. The reaction mixture
was stirred for 30 min after the addition of K2CO3 (50 mg) and
then washed with saturated NaHCO3 solution. The organic layer
was separated and dried over MgSO4. The organic solvent was
removed under reduced pressure. The crude product was purified
by column chromatography on silica gel with pentane–diethyl
ether (5 : 1) to obtain the THP ethers as colourless liquids.
Preparation of [D7]-5-methylhexanal 5
Similar to the method described by Corey and Suggs30
a
solution of [D7]-5-methylhexan-1-ol (238 mg, 1.93 mmol) in dry
dichloromethane (5.0 cm3) was added to a suspension of PCC
(619 mg, 2.88 mmol) in dry dichloromethane (5.0 cm3). The
reaction mixture was stirred at room temperature for 3 h and
then quenched by the addition of water (20 cm3). The aqueous
layer was separated and extracted with diethyl ether (3 × 20 cm3).
The combined extracts were dried with MgSO4 and the organic
solvents were removed under reduced pressure. The residue was
purified by column chromatography on silica gel with pentane–
diethyl ether (5 : 1) to give 5 (124 mg, 53%) as a colourless
liquid. Due to its instability against oxidation this aldehyde was
immediately used in the next step. TLC (pentane–diethyl ether =
5 : 1): RF 0.79; GC: I 861; m/z (EI) 102 (43%), 93 (25), 85 (32),
78 (71), 71 (33), 59 (82), 50 (73) and 46 (100).
2-(4-Bromobutyloxy)oxane 3. Yield: 3.18 g (91%); TLC
(pentane–diethyl ether = 5 : 1): RF 0.62; GC: I 1479;
dH(200 MHz; CDCl3; Me4Si) 1.52–1.63 (2 H, m, CH2), 1.65–
1.85 (2 H, m, CH2), 1.91–2.05 (2 H, m, CH2), 3.37–3.56 (2 H, m,
CH2) and 4.56–4.60 (1 H, m, CH); dC(50 MHz; CDCl3; Me4Si)
19.6 (CH2), 25.4 (CH2), 28.3 (CH2), 29.8 (CH2), 30.7 (CH2), 33.7
(CH2), 62.3 (CH2), 66.4 (CH2) and 98.8 (CH); m/z (EI) 237 (8),
235 (8), 137 (62), 135 (64), 101 (2), 85 (100), 67 (10), 55 (70) and
41 (35).
2-(10-Bromodecyloxy)oxane. Yield: 1.08 g (80%); TLC
(pentane–diethyl ether = 20 : 1): RF 0.15; GC: I 2135;
dH(200 MHz; CDCl3; Me4Si) 1.20–1.92 (22 H, m, 11 × CH2),
3.32–3.55 (2 H, m, CH2), 3.41 (2 H, t, J 6.8, CH2), 3.67–3.93
(2 H, m, CH2) and 4.56–4.59 (1 H, m, CH); dC(50 MHz; CDCl3;
Me4Si) 19.7 (CH2), 25.5 (CH2), 26.2 (CH2), 28.1 (CH2), 28.7
(CH2), 29.3 (CH2), 29.4 (CH2), 29.4 (CH2), 29.7 (CH2), 30.7
(CH2), 32.8 (CH2), 33.9 (CH2), 62.3 (CH2), 67.6 (CH2) and 98.8
(CH); m/z (EI) 321 (4%), 319 (4), 249 (1), 247 (1), 150 (2), 148
(2), 137 (2), 135 (2), 115 (2), 101 (9), 85 (100), 69 (10), 55 (22)
and 41 (22).
Preparation of (10-hydroxydecyl)triphenylphosphonium
bromide 6
A solution of 2-(10-bromodecyloxy)oxane (1.09 g, 3.38 mmol)
and triphenylphosphane (885 mg, 3.38 mmol) in acetonitrile
(20 cm3) was heated under reflux for 48 h. The reaction mixture
was concentrated in vacuo and the residue was purified by
column chromatography on silica gel with dichloromethane–
methanol (10 : 1) to yield 6 (1.20 g, 71%) as a colourless
solid. TLC (dichloromethane–methanol = 10 : 1): RF 0.38;
dH(200 MHz; CDCl3; Me4Si) 1.10–1.40 (10 H, m, 5 × CH2),
1.49–1.61 (6 H, m, 3 × CH2), 2.53 (1 H, br s, OH), 3.55–3.75
(2 H, m, CH2), 3.59 (2 H, t, J 6.6, CH2) and 7.73–7.87 (15 H, m,
15 × CH); dC(50 MHz; CDCl3; Me4Si) 22.5 (d, 3JP,C 4.4, CH2),
Preparation of [D7]-2-(5-methylhexyloxy)oxane 4
According to the method of Tamura and Kochi,28 a flame dried
reaction vessel was charged with Mg (158 mg, 6.50 mmol) and
dry THF (0.5 cm3) in a nitrogen atmosphere. A solution of
[D7]isopropyl bromide (845 mg, 6.50 mmol) in THF (2.5 cm3)
was added dropwise during 15 min. The reaction mixture was
stirred for an additional 30 min. After formation of the Grignard
reagent the mixture was cooled down to −78 ◦C, a solution of
3 (1.66 g, 7.00 mmol) in dry THF (25 cm3) and a solution of
Li2[CuCl4] in THF (0.7 cm3, 0.1 mol dm−3, 0.7 mmol) were added
dropwise. The reaction mixture was stirred overnight, allowed
to warm up slowly to room temperature and then quenched
by the addition of 2 N HCl (20 cm3). The aqueous layer was
extracted with diethyl ether (3 × 50 cm3). The combined organic
layers were dried with MgSO4 and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
with pentane–diethyl ether (20 : 1), and yielded 4 (700 mg, 52%)
as a colourless liquid. TLC (pentane–diethyl ether = 2 : 1): RF
0.37; GC: I 1373; dH(200 MHz; CDCl3; Me4Si) 1.15–1.44 (4 H,
m, 2 × CH2), 1.51–1.85 (8 H, m, 4 × CH2), 3.33–3.55 (2 H, m,
CH2), 3.68–3.93 (2 H, m, CH2) and 4.56–4.60 (1 H, m, CH);
dC(50 MHz; CDCl3; Me4Si) 19.6 (CH2), 23.9 (CH2), 25.5 (CH2),
29.9 (CH2), 30.7 (CH2), 38.5 (CH2), 62.3 (CH2), 67.6 (CH2) and
98.8 (CH); m/z (EI) 207 (1%), 206 (3), 134 (2), 115 (3), 101 (6),
85 (100), 67 (7), 56 (15) and 41 (15).
1
22.7 (d, JP,C 49.9, CH2), 25.6 (CH2), 28.9 (CH2), 28.9 (CH2),
2
29.1 (CH2), 29.1 (CH2), 30.3 (d, JP,C 15.5, CH2), 32.6 (CH2),
1
2
62.5 (CH2), 118.2 (d, JP,C 85.8, C), 130.5 (d, JP,C 12.3, CH),
133.5 (d, 3JP,C 9.9, CH) and 135.1 (d, 4JP,C 2.9, CH).
Preparation of [D7]-(Z)-15-methylhexadec-10-en-1-ol 7
As described by Martinez and Ruiz,32 a solution of BuLi in
hexane (3.0 cm3, 1.6 mol dm−3, 4.8 mmol) was added dropwise
to a suspension of 6 (1.20 g, 2.4 mmol) in dry THF (20 cm3). The
reaction mixture was stirred for 30 min. During this time a dark
red ylide was formed. A solution of 5 (97 mg, 0.80 mmol) in dry
THF (3.0 cm3) was added. The reaction mixture was stirred at
room temperature for 2 h and then quenched by the addition of
2 N HCl. The aqueous layer was separated and extracted with
diethyl ether (3 × 50 cm3). The combined extracts were dried with
MgSO4 and concentrated under reduced pressure. Compound
7 (94 mg, 45%) was obtained by column chromatography on
silica gel with pentane–diethyl ether (5 : 1) as a colourless liquid.
Only minor amounts (<5%) of the E-isomer were detected by
1H NMR.
TLC (pentane–diethyl ether = 5 : 1): RF 0.15; GC: I 1933;
dH(200 MHz; CDCl3; Me4Si) 1.13–1.41 (14 H, m, 7 × CH2),
1.50–1.61 (4 H, m, 2 × CH2), 1.86 (1 H, br s, OH), 1.94–1.99
(4 H, m, 2 × CH2), 3.62 (2 H, t, J 6.6, CH2) and 5.27–5.43 (2 H, m,
2 × CH); dC(50 MHz; CDCl3; Me4Si) 25.7 (CH2), 27.2 (CH2),
27.4 (CH2), 27.5 (CH2), 29.2 (CH2), 29.4 (CH2), 29.4 (CH2),
29.4 (CH2), 29.5 (CH2), 29.7 (CH2), 32.7 (CH2), 62.9 (CH2),
129.8 (CH) and 129.9 (CH); m/z (EI) 261 (3), 243 (16), 215 (5),
Preparation of [D7]-5-methylhexan-1-ol
A solution of 4 (623 mg, 3.01 mmol) and p-toluenesulfonic acid
(58 mg, 0.30 mmol) in dry methanol (20 cm3) was stirred for
30 min at room temperature, and then the solvent was removed
under reduced pressure. The residue was diluted with water and
extracted with diethyl ether (3 × 50 cm3). The combined organic
layers were dried with MgSO4 and concentrated. Purification
2 8 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 2 4 – 2 8 3 1

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187 (3), 152 (3), 138 (9), 124 (18), 110 (31), 96 (74), 82 (100), 75
(34), 67 (86), 55 (95) and 41 (72).
10 C. J. Dutton, S. P. Gibson, A. C. Goudie, K. S. Holdom, M. S. Pacey,
J. C. Ruddock, J. D. Bu’Lock and M. K. Richards, J. Antibiot., 1991,
44, 357–365.
11 T. Mahmud, S. C. Wenzel, E. Wan, K. W. Wen, H. B. Bode, N.
Gaitatzis and R. Mu¨ller, ChemBioChem., 2005, 6, 322–330.
12 T. Mahmud, H. B. Bode, B. Silakowski, R. M. Kroppenstedt, M.
Xu, S. Nordhoff, G. Ho¨fle and R. Mu¨ller, J. Biol. Chem., 2002, 277,
32768–32774.
13 J. C. Ware and M. Dworkin, J. Bacteriol., 1973, 115, 253–261.
14 J. Schro¨der and H. Reichenbach, Arch. Microbiol., 1970, 71, 384–390.
15 G. Bartholomeusz, Y. Zhu and J. Downard, J. Bacteriol., 1998, 180,
5269–5272.
16 D. B. Kearns, A. Vernot, P. J. Bonner, B. Stevens, G.-J. Boons and
L. J. Shimkets, Proc. Natl. Acad. Sci. USA, 2001, 98, 13990–13994.
17 H. B. Bode, J. Dickschat, R. M. Kroppenstedt, S. Schulz and R.
Mu¨ller, J. Am. Chem. Soc., 2005, 127, 532–533.
Preparation of [D7]-15-methylhexadecan-1-ol
Pd/C (21 mg, 10% Pd, 0.02 mmol) was added to a solution of
7 (94 mg, 0.36 mmol) in diethyl ether (5.0 cm3). The reaction
mixture was stirred in an H2 atmosphere (10 N cm−2) for 20 min.
Pd/C was filtered of and the resulting solution was concentrated.
The product was purified by column chromatography on silica
gel with pentane–diethyl ether (3 : 1) to obtain [D7]-15-
methylhexadecan-1-ol (74 mg, 78%) as a colourless solid.
TLC (pentane–diethyl ether = 3 : 1): RF 0.32; GC: I 1951;
dH(200 MHz; CDCl3; Me4Si) 1.15–1.40 (24 H, m, 12 × CH2),
1.50–1.64 (2 H, m, CH2), 1.66 (1 H, br s, OH) and 3.63 (2 H, t, J
6.5, CH2); dC(50 MHz; CDCl3; Me4Si) 25.7 (CH2), 27.3 (CH2),
29.4 (2 × CH2), 29.6 (3 × CH2), 29.7 (4 × CH2), 29.9 (CH2),
32.8 (CH2) and 63.0 (CH2); m/z (EI) 245 (8%), 217 (14), 189 (5),
157 (4), 140 (4), 125 (8), 111 (20), 97 (52), 83 (82), 69 (79), 55
(100) and 41 (63).
18 F. W. McLafferty and F. Turecˇek, Interpretation von Massenspektren,
Spektrum Akademischer Verlag, Heidelberg, Berlin, Oxford, 1995.
19 R. Ryhage and E. Stenhagen, Ark. Kemi, 1960, 15, 291–315.
20 J. S. Dickschat, S. C. Wenzel, H. B. Bode, R. Mu¨ller and S. Schulz,
ChemBioChem., 2004, 4, 778–787.
21 S. Schulz, Lipids, 2001, 36, 637–647.
22 E. Fautz, G. Rosenfelder and L. Grotjahn, J. Bacteriol., 1979, 140,
852–858.
Preparation of [D7]-15-methylhexadecanoic acid 8
23 S. Yamanaka, R. Fudo, A. Kawaguchi and K. Komagata, J. Gen.
Appl. Microbiol., 1988, 34, 57–66.
According to the method of Corey and Schmidt33 PDC (368 mg,
0.98 mmol) was suspended in dry DMF (1.0 cm3). A solution of
[D7]-15-methylhexadecan-1-ol (74 mg, 0.28 mmol) in dry DMF
(3.0 cm3) was added to this suspension. The reaction mixture was
stirred for 12 h at room temperature and then water (20 cm3)
was added. The aqueous layer was separated and extracted
with diethyl ether (3 × 50 cm3). The combined organic layers
were dried with MgSO4 and concentrated to dryness. The crude
product was purified by column chromatography on silica gel
with pentane–diethyl ether (3 : 1) to yield 8 (52 mg, 67%) as
a colourless solid. For GC–MS analysis a small sample was
transformed into its trimethylsilyl ester with MSTFA.50 TLC
(pentane–diethyl ether = 3 : 1): RF 0.51; dH(200 MHz; CDCl3;
Me4Si) 1.10–1.40 (20 H, m, 10 × CH2), 1.55–1.72 (4 H, m, 2 ×
CH2) and 2.35 (2 H, t, J 7.5, CH2); dC(50 MHz; CDCl3; Me4Si)
24.7 (CH2), 27.4 (CH2), 29.0 (CH2), 29.2 (CH2), 29.4 (CH2), 29.6
(CH2), 29.6 (2 × CH2), 29.9 (CH2), 34.0 (CH2), 38.8 (CH2) and
179.9 (CO); m/z (EI, MSTFA) 349 (14%), 334 (100), 320 (1),
306 (5), 290 (4), 201 (6), 185 (3), 145 (20), 132 (27), 129 (27), 117
(50), 75 (33), 73 (38) and 50 (9).
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